Currently the pace of change in techniques and tools for discovery of biologically active molecules is increasing with the ability of combinatorial chemistry and multi-parallel synthesis (MPS) to rapidly provide large numbers of diverse molecules to test for biological activity. In addition the mapping and sequencing of the genomes of many plants, animals and parasites, including the human genome, is already providing a growing number of new targets which may be used in biological tests. In the future it is to be expected that the number of biological targets is to grow even further. It is estimated that in the last 100 years of research only 400 human drug targets have been discovered whilst the human genome project when completed at the scheduled time of 2005 will have sequenced at least 100,000 genes, many of which will code for important biological targets for drug therapy.
However, synthetic techniques such as MPS and combinatorial chemistry provide relatively small sample sizes, for example in the microgram range. The limited sample sizes currently produced are not large enough to supply more than a few biological tests before the supply is exhausted. Therefore, resynthesis is required in order to restock the chemical library.
Currently there is an enormous range of in vitro assay techniques used for biological tests. Generally an in vitro biological test involves exposing the isolated biological target to the compound under investigation and measuring interference with the normal binding of the biological target to its ligand. Such tests are typically run on standard 96 well plates and require a minimum sample size of biological target of around 0.1 ml and a minimum amount of a sample size of compound of around 1 μg.
As well as supplies of the test compound running out, supplies of biological target molecules also may be quickly depleted. It is an expensive and tedious task to have to express, isolate and purify the biological target from the biological source.
Therefore, there is a need to find simple, sensitive, high-throughput approaches to the identification of compounds inhibiting ligands binding to biologically important target molecules for use in the pharmaceutical and agrochemical industry. Such a system should use small sample sizes and be amenable to operation by a machine. In particular, miniaturised approaches operating on the picolitre/nanolitre/microlitre scale are particularly desirable, because of the large cost savings and potential for very high throughput. So far there are very few approaches that will work satisfactorily at this scale. One such approach, fluorescence correlation spectroscopy, which is based on differences in diffusion coefficient measured by fluorescence in femtolitre interrogation volume is inherently slow because it measures only small numbers of molecules.
Microfabrication techniques are generally known in the art using tools developed by the semiconductor industry to miniturise electronics, and it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests. See, e.g., Ramsey, J. M. et al. (1995), “Microfabricated chemical measurement Systems,” Nature Medicine 1:1093-1096; and Harrison, D. J. et al. (1993), “Micromachining a minaturized capillary electrophoresis-based chemical analysis system on a chip,” Science 261:895-897.
Miniaturisation of laboratory techniques is not a simple matter of reducing their size. At small scales different effects become important, rendering some processes inefficient and others useless. It is difficult to replicate smaller versions of some devices because of material or process limitations. For these reasons it is necessary to develop new methods for performing common laboratory tasks on the microscale.
Devices made by micromachining planar substrates have been made and used for chemical separation, analysis, and sensing. See, e.g., Manz, A. et al. (1994), “Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis system,” J. Micromech. Microeng. 4: 257-265. In addition devices have been proposed for preparative, anayltical and diagnostic methods which bring two streams of fluid in laminar flow together which allows molecules to diffuse from one stream to the next, examples are proposed in WO9612541, WO9700442 and U.S. Pat. No. 5,716,852.